Adaptive Copy Number Evolution in Malaria Parasites

Citation: Nair S, Miller B, Barends M, Jaidee A, Patel J, et al. ( Adaptive Copy Number Evolution in Malaria Parasites Shalini Nair 0 Becky Miller 0 Marion Barends 0 Anchalee Jaidee 0 Jigar Patel 0 Mayfong Mayxay 0 Paul Newton 0 Fran cois Nosten 0 Michael T. Ferdig 0 Tim J. C. Anderson 0 Molly Przeworski, University of Chicago, United States of America 0 1 Southwest Foundation for Biomedical Research (SFBR) , San Antonio, Texas , United States of America, 2 Department of Biological Sciences, Eck Institute for Global Health, University of Notre Dame, Notre Dame, Indiana, United States of America, 3 Shoklo Malaria Research Unit (SMRU) , Mae Sot, Tak , Thailand , 4 Faculty of Tropical Medicine, Mahidol University , Bangkok, Thailand, 5 Roche NimbleGen , Inc., Madison, Wisconsin, United States of America, 6 Wellcome Trust-Mahosot Hospital-Oxford Tropical Medicine Research Collaboration, Mahosot Hospital , Vientiane , Lao People's Democratic Republic, 7 Department of Post Graduates and Research, Faculty of Medical Science, National University of Laos , Vientiane , Lao People's Democratic Republic, 8 Centre for Tropical Medicine and Vaccinology, Churchill Hospital , Oxford , United Kingdom Copy number polymorphism (CNP) is ubiquitous in eukaryotic genomes, but the degree to which this reflects the action of positive selection is poorly understood. The first gene in the Plasmodium folate biosynthesis pathway, GTP-cyclohydrolase I (gch1), shows extensive CNP. We provide compelling evidence that gch1 CNP is an adaptive consequence of selection by antifolate drugs, which target enzymes downstream in this pathway. (1) We compared gch1 CNP in parasites from Thailand (strong historical antifolate selection) with those from neighboring Laos (weak antifolate selection). Two percent of chromosomes had amplified copy number in Laos, while 72% carried multiple (2-11) copies in Thailand, and differentiation exceeded that observed at 73 synonymous SNPs. (2) We found five amplicon types containing one to greater than six genes and spanning 1 to .11 kb, consistent with parallel evolution and strong selection for this gene amplification. gch1 was the only gene occurring in all amplicons suggesting that this locus is the target of selection. (3) We observed reduced microsatellite variation and increased linkage disequilibrium (LD) in a 900-kb region flanking gch1 in parasites from Thailand, consistent with rapid recent spread of chromosomes carrying multiple copies of gch1. (4) We found that parasites bearing dhfr-164L, which causes high-level resistance to antifolate drugs, carry significantly (p = 0.00003) higher copy numbers of gch1 than parasites bearing 164I, indicating functional association between genes located on different chromosomes but linked in the same biochemical pathway. These results demonstrate that CNP at gch1 is adaptive and the associations with dhfr-164L strongly suggest a compensatory function. More generally, these data demonstrate how selection affects multiple enzymes in a single biochemical pathway, and suggest that investigation of structural variation may provide a fast-track to locating genes underlying adaptation. - Funding: This work was funded by grant from NIH (R01 AI075145 and AI48071) to TJCA. The molecular work at the Southwest Foundation for Biomedical Research was conducted in facilities constructed with support from Research Facilities Improvement Program grant C06 RR013556 from the National Center for Research Resources, NIH. The funders played no role in the design and conduct of the study, in the collection, analysis, and interpretation of the data, and in the preparation, review, or approval of the manuscript. Competing Interests: The authors have declared that no competing interests exist. A spate of studies over the past five years have described widespread copy number variation (CNP) within the genomes of humans, mice, Drosophila and other eukaryotes [15]. The existence of large regions of the genome that vary in copy number between individuals has lead to a reconsideration of the importance of structural variation for our understanding of genetic and phenotypic variation [6]. However, it is unclear whether CNP evolution is predominantly neutral, or whether positive or negative selection play significant roles in shaping the patterns observed [1]. The fact that CNPs tend to be enriched for particular gene classes, and for genes showing evidence for positive selection at the nucleotide level, strongly suggests the action of positive selection [7], although the alternative explanation of purifying selection against CNP in particular gene classes cannot be discounted. Furthermore, that CNPs explain ,20% of variance in transcript abundance in humans suggests that they have the potential to make a significant contribution to disease susceptibility and adaptive evolution [8]. However, despite these indirect lines of evidence for positive selection, adaptive copy number evolution has been demonstrated or hypothesized in only a few cases. In humans there are two notable examples. Gonzales et al [9] showed that protection from HIV is associated with CNP at the CCL3L1 gene. This CNP shows extreme geographical variation which further supports the action of selection by HIV (or, more likely, by an older human pathogen) [10]. Similarly, Perry et al [11] showed higher copy number of the amylase gene in populations with high starch diets. CNP is also widespread in the malaria parasite genome [12,13]. Malaria parasites are exposed to strong selection from the human immune response and treatment with antimalarial drugs. They have relatively small genomes (23 Mb) and haploid genetics, and can be grown and genetically manipulated in the laboratory, so provide a useful eukaryotic organism for investigating the functional role of CNP. One CNP on chromosome (chr.) 5 is known to underlie a multidrug resistance phenotype: chromosomes carrying this CNP Recent comparative genomic hybridization studies have revealed extensive copy number variation in eukaryotic genomes. The first gene in the Plasmodium folate biosynthesis pathway, GTP-cyclohydrolase I (gch1), shows extensive copy number polymorphism (CNP). We provide compelling evidence that gch1 CNP is adaptive and most likely results from selection by antifolate drugs, which target enzymes downstream in this pathway. Gch1 CNP shows extreme geographical differentiation; hitchhiking reduces diversity and increases LD in flanking sequence, indicating recent rapid spread within Thailand, while amplicon structure reveals multiple origins and parallel evolution. Furthermore, strong association between elevated copy number and a critical mutation dhfr-I164L that underlies high-level antifolate resistance indicates functional linkage and fitness epistasis between genes on different chromosomes. These data reveal hidden complexity in the evolutionary response to antifolate treatment and demonstrate that analysis of stru (...truncated)